Met tyrosine kinase is a high-affinity transmembrane receptor for the hepatocyte growth factor (HGF, Bottaro et al. (1991) Science 251:802-804). Met was cloned, named (Cooper et al. (1984) 311:29-33) and identified as an oncogene (Park et al. (1986) Cell 45:895-904). When deregulated by overexpression or mutations, Met receptor tyrosine kinase leads to tumor growth and invasion (Cristiani et al. (2005) Biochem. 44:14110-14119). Stimulation of Met by the ligand HGF, also known as Scatter Factor, initiates numerous physiological processes, including cell proliferation, scattering, morphogenic differentiation, angiogenesis, wound healing, tissue regeneration, and embryological development (Parr et al. (2004) Clin. Cancer Res. 10(1, Pt. 1) 202-211; Comoglio et al. (2002) J. Clin. Invest. 109:857-862; Maulik et al. (2002) Cytokine Growth Factor Reviews 13:41-59; Hecht et al. (2004) Cancer Res. 64(17):6109-6118). Receptor c-Met is rapidly internalized via clathrin-coated vesicles and traffics through an early endosomal compartment after hepatocyte growth factor stimulation. c-Met accumulates progressively in perinuclear compartments, which in part include the Golgi (Kermorgant et al. (2003) J. of Biol. Chem. 278(31):28921-28929).
The phenomena of: deregulation or dysregulation of Met and/or HGF; Met overexpression; and Met mutations are implicated in uncontrolled cell proliferation and survival, and play a key role in early-stage tumorigenesis, invasive growth of cancer cells, and metastasis (Danilkovitch-Miagkova et al. (2002) J. Clin. Invest. 109(7):863-867; Di Renzo et al. (1994) Int. J. Cancer 58:658-662; Matsumoto et al. (1994) J. Biol. Chem. 269:31807-31813; Tusolino et al. (1998) J. Cell Biol. 142:1145-1156; Jeffers et al. (1996) Mol. Cell. Biol. 16:1115-1125; Wong et al. (2004) Exper. Cell Res. 299(1):248-256; Konda et al. (2004) Jour. of Urology 171(6), Pt. 1:2166-2170; Heideman et al. (2004) J. Gene Med. 6(3):317-327; Ma et al. (2003) Cancer Res. 63(19):6272-6281; Maulik et al. (2002) Clin. Cancer Res. 8:620-627), making Met an important target for anticancer drug development (Cohen, P. (2002) Nat. Rev. Drug Discovery 1:309-315). Overexpression of Met and HGF is associated with poor prognosis.
Recent data demonstrating the suppression of cancer cell proliferation, survival, and invasion upon inhibition of Met binding to HGF and Met receptor dimerization (Furge et al. (2001) Proc. Natl. Acad. Sci. USA 98:10722-10727; Michieli et al. (2004) Cancer Cell 6:61-73) confirm the relevance of Met in neoplasia and provide further proof of concept for the development of small-molecule compounds for antineoplastic therapy, e.g. against multiple myeloma (Hov et al. (2004) Clin. Cancer Res. 10(19):6686-6694). Inhibition of Met results in slowing tumor growth in tumor xenograft mouse models. Antibodies specific for c-Met have been expressed to block binding of HGF to c-Met (US 2005/0037431; US 2004/0166544).
Protein kinases (PK) are enzymes that catalyze the phosphorylation of hydroxy groups on tyrosine, serine and threonine residues of proteins by transfer of the terminal (gamma) phosphate from ATP. Through signal transduction pathways, these enzymes modulate cell growth, differentiation and proliferation, i.e., virtually all aspects of cell life in one way or another depend on PK activity. Furthermore, abnormal PK activity has been related to a host of disorders, ranging from relatively non-life threatening diseases such as psoriasis to extremely virulent diseases such as glioblastoma (brain cancer). Protein kinases include two classes; protein tyrosine kinases (PTK) and serine-threonine kinases (STK).
One of the prime aspects of PTK activity is their involvement with growth factor receptors which are cell-surface proteins. When bound by a growth factor ligand, growth factor receptors are converted to an active form which interacts with proteins on the inner surface of a cell membrane. This leads to phosphorylation on tyrosine residues of the receptor and other proteins and to the formation inside the cell of complexes with a variety of cytoplasmic signaling molecules that, in turn, effect numerous cellular responses such as cell division (proliferation), cell differentiation, cell growth, expression of metabolic effects to the extracellular microenvironment, etc. For a more complete discussion, see Schlessinger and Ullrich, (1992) Neuron 9:303-391.
Growth factor receptors with PTK activity are known as receptor tyrosine kinases (RTK, Plowman et al. (1994) DN&P, 7(6):334-339), which comprise a large family of transmembrane receptors with diverse biological activity. At present, at least nineteen (19) distinct subfamilies of RTK have been identified. An example of these is the subfamily designated the “HER” RTK, which include EGFR (epithelial growth factor receptor), HER2, HER3 and HER4. These RTK consist of an extracellular glycosylated ligand binding domain, a transmembrane domain and an intracellular cytoplasmic catalytic domain that can phosphorylate tyrosine residues on proteins. Another RTK subfamily consists of insulin receptor (IR), insulin-like growth factor I receptor (IGF-1R) and insulin receptor related, receptor (IRR). IR and, IGF-1R interact with insulin, IGF-I and IGF-II to form a heterotetramer of two entirely extracellular glycosylated alpha subunits and two beta subunits which cross the cell membrane and which contain the tyrosine kinase domain. A third RTK subfamily is referred to as the platelet derived growth factor receptor (PDGFR) group, which includes PDGFR-alpha, PDGFR-beta, CSFIR, c-kit and c-fms. These receptors consist of glycosylated extracellular domains composed of variable numbers of immunoglobin-like loops and an intracellular domain wherein the tyrosine kinase domain is interrupted by unrelated amino acid sequences. Another group which, because of its similarity to the PDGFR subfamily, is sometimes subsumed into the later group is the fetus liver kinase (flk) receptor subfamily. This group is believed to be made up of kinase insert domain-receptor fetal liver kinase-1 (KDR/FLK-1), flk-1R, flk-4 and fms-like tyrosine kinase 1 (flt-1). Another member of the tyrosine kinase growth factor receptor family is the fibroblast growth factor (“FGF”) receptor subgroup. This group consists of four receptors, FGFR1-4, and seven ligands, FGF1-7. While not yet well defined, it appears that these receptors consist of a glycosylated extracellular domain containing a variable number of immunoglobin-like loops and an intracellular domain in which the tyrosine kinase sequence is interrupted by regions of unrelated amino acid sequences. Still another member of the tyrosine kinase growth factor receptor family is the vascular endothelial growth factor (VEGF) receptor subgroup. VEGF is a dimeric glycoprotein similar to PDGF but has different biological functions and target cell specificity in vivo. In particular, VEGF is presently thought to play an essential role is vasculogenesis and angiogenesis.
Met is still another member of the tyrosine kinase growth factor receptor family, and often referred to as c-Met or human hepatocyte growth factor receptor tyrosine kinase (hHGFR). The expression of c-Met is thought to play a role in primary tumor growth and metastasis (Kim et al. Clin. Cancer Res. (2003) 9(14):5161-5170).
Modulation of the HGF/c-Met signaling pathway may be effected by regulating binding of HGF beta chain to cMet. In particular embodiments, the zymogen-like form of HGF beta mutant was shown to bind Met with 14-fold lower affinity than the wild-type serine protease-like form, suggesting optimal interactions result from conformational changes upon cleavage of the single-chain form (US 2005/0037431). Extensive mutagenesis of the HGF beta region corresponding to the active site and activation domain of serine proteases showed that 17 of the 38 purified two-chain HGF mutants resulted in impaired cell migration or Met phosphorylation but no loss in Met binding. However, reduced biological activities were well correlated with reduced Met binding of corresponding mutants of HGF beta itself in assays eliminating dominant alpha-chain binding contributions.
Protein-tyrosine kinases (PTK) are critical components of signaling pathways that control cellular proliferation and differentiation. PTK are subdivided into two large families, receptor tyrosine kinases (RTK) and non-receptor tyrosine kinases (NRTK). RTK span the plasma membrane and contain an extra-cellular domain, which binds ligand, and an intracellular portion, which possesses catalytic activity and regulatory sequences. Most RTK, like the hepatocyte growth factor receptor c-met, possess a single polypeptide chain and are monomeric in the absence of a ligand. Ligand binding to the extracellular portion of RTK, dimerizes monomeric receptors, resulting in autophosphorylation of specific tyrosine residues in the cytoplasmic portion (for review see: Blume-Jensen, P., and Hunter, T., Nature (2001) 411:355-365; Hubbard, S. R., et al., J. Biol. Chem. 273 (1998) 11987-11990; Zwick, E., et al., Trends Mol. Med. (2002) 8:17-23). In general, tyrosine autophosphorylation either stimulates the intrinsic catalytic kinase activity of the receptor or generates recruitment sites for downstream signaling proteins containing phosphotyrosine-recognition domains, such as the Src homology 2 (SH2) domain or the phosphotyrosine-binding (PTB) domain.
PTK have become primary targets for the development of novel therapeutics designed to block cancer cell proliferation, metastasis, and angiogenesis and promote apoptosis. The strategy that has progressed farthest in clinical development is the use of monoclonal antibodies to target growth factor receptor tyrosine kinases. The use of small molecule tyrosine kinase inhibitors however could have significant theoretical advantages over monoclonal antibodies. Small molecule inhibitors could have better tissue penetration, could have activity against intracellular targets and mutated targets and could be designed to have oral bioavailability. Several lead compounds have shown promising activity against such targets as the EGFR, the vascular endothelial cell growth factor receptor and bcr-abl. The hepatocyte growth factor receptor c-Met was first identified as an activated oncogene in an N-methyl-N′-nitrosoguanidinic treated human osteogenic sarcoma cell line (MUNG-HOS) by its ability to transform NIH 3T3 mouse fibroblasts. The receptor encoded by the c-Met protooncogene (located on chromosome 7) is a two-chain protein composed of 50 kDa (alpha) chain disulfide linked to a 145 kDa (beta) chain in an alpha-beta complex of 190 kDa. The alpha-chain is exposed at the cell surface while the beta chain spans the cell membrane and possesses an intracellular tyrosine kinase domain. The presence of this intracellular tyrosine kinase domain groups c-Met as a member of the receptor tyrosine kinase (RTK) family of cell surface molecules.
Much evidence supports the role of HGF as a regulator of carcinogenesis, cancer invasion and metastasis (for review see: Herynk, M. H., and Radinsky, R. (2000) In Vivo 14:587-596; Jiang et al. (1999) Crit. Rev. Oncol. Hematol. 29:209-248; Longati (2001) Curr. Drug Targets 2:41-55; Maulik et al., (2002) Cytokine Growth Factor Rev. 13:41-59; Parr, C., and Jiang, W. G., (2001) Histol. Histopathol. 16:251-268). HGF binds to and induces tyrosine phosphorylation of the mature c-met receptor beta-chain. Such events are thought to promote binding of intracellular signaling proteins containing src homology (SH) regions such as PLC-gamma, Ras-GAP, PI-3 kinase pp60c-src and the GRB-2 Socs complex to the activated receptor. Each SH2-containing protein may activate a different subset of signaling phosphopeptides, thus eliciting different responses within the cell. c-Met mutations have been well-described in hereditary and sporadic human papillary renal carcinomas and have been reported in ovarian cancer, childhood hepatocellular carcinoma, metastatic head and neck squamous cell carcinomas, and gastric cancer. c-Met is also over-expressed in both non-small cell lung cancer and small cell lung cancer cells, in lung, breast, colon and prostate tumors (Herynk et al. (2003) Cancer Res. 63(11):2990-2996; Maulik et al. (2002) Clin. Cancer Res. 8:620-627). Since c-Met appears to play an important role in oncogenesis of a variety of tumors, various inhibition strategies have been employed to therapeutically target this receptor tyrosine kinase. The usefulness of inhibiting the protein-tyrosine kinase c-Met for inhibiting tumor growth and invasion has been shown in many well documented preclinical experiments (Abounader et al. (1999) J. Natl. Cancer Inst. 91:1548-1556; Laterra et al. (1997) Lab. Invest. 76:565-577; Tomioka, D. (2001) Cancer Res. 61:7518-7524; Wang et al. (2001) J. Cell Biology 153:1023-1033).
cMet inhibitors have been reported (US 2004/0242603; US 2004/0110758; US 2005/0009845; WO 2003/000660; WO 98/007695; U.S. Pat. No. 5,792,783; U.S. Pat. No. 5,834,504; U.S. Pat. No. 5,880,141; US 2003/0125370; U.S. Pat. No. 6,599,902; WO 2005/030140; WO 2005/070891; US 2004/0198750; U.S. Pat. No. 6,790,852; WO 2003/087026; U.S. Pat. No. 6,790,852; WO 2003/097641; U.S. Pat. No. 6,297,238; WO 2005/005378; WO-2004/076412; WO 2005/004808; WO 2005/010005; US 2005/0009840; WO 2005/121125). PHA-665752 is a small molecule, ATP-competitive, active-site inhibitor of the catalytic activity of c-Met, as well as phenotypes such as cell growth, cell motility, invasion, and morphology of a variety of tumor cells (Ma et al. (2005) Clin. Cancer Res. 11:2312-2319; Christensen et al. (2003) Cancer Res. 63:7345-7355).